The goal of this project is to investigate the role of free radical formation in cerebral ischemia and to develop therapies that may reduce their deleterious side effects. Biochemical changes that accompany cerebral ischemia include formation of free radicals such as the superoxide anion O2, and these highly reactive substances may be an important cause of neuronal damage. Studies showing efficacy with pretreatment with free radicals scavengers have been conflicting and when protection was demonstrated, it was attributed to action at the level of the vascular endothelium and it was unclear whether free radicals were generated in dying neurons as well. The hypotheses to be tested in this project are: free radical formation plays a role in the ischemic cascade, occurs intracellularly; individual contributions of hypoxia and substrate deprivation, the major determinants of ischemia both play a role in free radical formation, and that free radical scavengers if taken up intracellularly will protect from ischemic damage. These hypotheses will be tested by first using a tissue culture model of neuronal ischemia where both hypoxia and substrate deprivation will be tested for individually and in combination. Intracellular free radical formation accumulation will be demonstrated by measuring reduction of nitroblue tetrazolium (NBT) and the oxidation of 21, 71-dichlorofluorescen to dichlorfluorescein. Neuronal survival will be measured by uptake of tryphan blue. Superoxide dismutase (a free radical scavenger) modified by entrapping in liposomes as well as a copper complex will be studied to see if they offer neuronal protection. Finally, those agent(s) that show promise in tissue culture will be studied in an animal model of transient forebrain ischemia. Outcome will be measured functionally (maze testing), histologically and biochemically (ATP, PCr and glucose). Ischemic brain edema (as measured by water content) will also be evaluated. Improvement in outcome in this animal model as related to free radical damage will be tested by measuring superoxide dismutase activity, free radical formation by NBT, and lipid peroxidation as measured by the thiobarbituric acid test.